Synthesis, characterization, and reaction pathways for the formation of a GMP adduct of a cytotoxic thiocyanato ruthenium arene complex.

J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 DOI 10.1007/s00775-009-0537-1 POSTER PRESENTATION Bioenergetics P001 Low temperature photolysis study of NO, NO/CO and HNO complexes in cytochrome bo3 from Escherichia coli Takahiro Hayashi1, Myat T. Lin2, Robert B. Gennis2, Pierre Moënne-Loccoz1 1 Department of Science and Engineering, School of Medicine, Oregon Health and Science University, 20,000 NW Walker Road, Beaverton, OR 97006, USA. 2 Department of Biochemistry, University of Illinois, 600 S Mathews Avenue, Urbana, IL 61801, USA. hayashi@ebs.ogi.edu Bacterial heme–copper terminal oxidases react with NO to form heme–nitrosyl complexes, which, in some of these enzymes, can further react with a second NO to produce N2O (2NO + 2e- + 2H+ ? N2O + H2O). It is unknown whether the formation of CuB–NO is an obligate step of this catalytic reaction. Here, we present low-temperature photolysis experiments on NO, NO/CO and HNO complexes of reduced cytochrome bo3, which is a quinol oxidase with NO reductase activity. Upon illumination of bo3–NO, NO dissociates from heme-o3 to dock into a proteinaceous pocket and shows m(NO) at 1,863 cm-1. When the same experiment is carried out in the presence of CO, an [o3–NOOC–CuB] tertiary complex is formed at the active site. We also examined the reaction of bo3 with Angeli’s salt, a HNO donor, and characterized an o3–HNO complex. These data suggest that in bo3, NO is activated at the heme–o3 and that the role of the CuB is limited to promoting the formation of a heme iron-hyponitrite species through electrostatic interaction. ν(NO)o3=1610 cm-1 ν(NO)o3=1610 cm-1 ν(NO)free=1863 cm ν(NO)free=1863 cm-1 ν(N(H)O)o3=1380 cm -1 -1 ν(CO)CuB=2057 cm-1 photochemically excited and oxidized. Following electronic excitation and oxidation of the Re(I) sensitizer, electron transfer from a distant Ru(II) donor, which is attached to another cysteine residue positioned outside the vesicle when folded, through intermediate trytophans to the Re(I)* acceptor will be monitored by transient absorption. P003 Hydrogen bond network around the non-heme iron in photosystem II Ryouta Takahashi1, Miwa Sugiura2, Alain Boussac3, Takumi Noguchi1 1 Institute of Materials Science, University of Tsukuba, Tsukuba 305-8573, Japan. 2 Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan. 3 iBiTec-S, URA CNRS 2096, CEA Saclay 91191 Gif sur Yvette, France. s-takahashi@ims.tsukuba.ac.jp In photosystem II (PSII), a non-heme iron which is located between the primary (QA) and secondary (QB) quinone acceptors controls the electron transfer from QA to QB. The recent X-ray structures showed that D1-Y246, D2-Y244 and D1-E244 are located near the bicarbonate molecule coordinating to the non-heme iron. However, the details of their H-bond structures and the roles in the quinone reactions have not yet been clarified. In this study, we have studied the molecular interactions around the non-heme iron by means of Fourier transform infrared (FTIR) spectroscopy. A light-induced Fe2+/Fe3+ difference spectrum in the PS II core complexes showed peaks at 1,256/1,232 cm-1, which downshifted by 16 cm-1 upon labeling of Tyr side chains with 4-13C-Tyr. These peaks were assigned to the COH vibrations of a Tyr side chain(s), which may be attributed to D1Y246 and/or D2-Y244. The C=O/COH bands of bicarbonate and the C=O band of a COOH group were also detected. From these data, it is proposed that D1-Y246, D2-Y244, and D1-E244 interact with the bicarbonate to form an H-bond network around the non-heme iron, which can be involved in a proton transfer pathway to QB (Fig. 1). P002 Multi-step electron tunneling in a transmembrane protein Bert T. Lai, Jay R. Winkler, Harry B. Gray Beckman Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA. bert@caltech.edu Multi-step electron tunneling has been demonstrated experimentally using a rhenium-labeled metalloprotein in solution. However, in natural redox systems, such as those involved in photosynthesis and respiration, multi-step electron tunneling must occur across a membrane. The research here involves studying electron tunneling through a transmembrane protein, namely outer membrane protein A (OmpA). A rhenium sensitizer complex will be covalently bound to a cysteine residue folded inside a small unilamellar vesicle. It will be Fig. 1 H-bond network around the non-heme iron 123 S102 P004 FTIR Detection of the DOD bending vibrations of water molecules involved in the photosynthetic water oxidation Hiroyuki Suzuki1, Miwa Sugiura2, Takumi Noguchi1 1 Institute of Materials Science, University of Tsukuba, Ibaraki 305-8573, Japan. 2 Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan. hisuzuki@ims.tsukuba.ac.jp Photosynthetic water oxidation takes place at the water oxidizing center (WOC) in photosystem II (PSII). The reaction proceeds through a light-driven cycle of five intermediates called Sn state (n = 0–4). During the S-state cycle, two water molecules are oxidized into one O2 molecule and four protons. To clarify the mechanism of water oxidation, it is of utmost importance to detect water molecules in WOC and monitor their reactions at the molecular level. In previous studies [1, 2], we detected water molecules as OH stretching vibrations using Fourier transform infrared (FTIR) spectroscopy. However, the OH vibrations of strongly H-bonded water have been difficult to detect because of significant band broadening. In this study, we have detected the DOD bending vibrations of heavy water involved in photosynthetic water oxidation [3]. Flash-induced FTIR difference spectra during the S-state cycle were measured with PSII core films of T. elongatus moderately deuterated with D16 2 O or 18 16 D18 2 O. The D2 O-minus-D2 O double difference spectra at individual S-state transitions (S1 ? S2, S2 ? S3, S3 ? S0, and S0 ? S1) showed six to eight DOD peaks in the 1,150–1,250 cm-1 region, indicating that at least two water molecules, not in any deprotonated forms, participate in the reaction at each transition throughout the cycle. The negative bands at *1,240 cm-1 in the S2 ? S3 and S3 ? S0 transitions did not have corresponding counter bands in other transitions. This result suggests that substrate water molecules are inserted into the catalytic site in the S2 ? S3 and S3 ? S0 transitions. References 1. Noguchi T, Sugiura M (2000) Biochemistry 39:10943-10949 2. Noguchi T, Sugiura M (2002) Biochemistry 41:15706–15712 3. Suzuki H, Sugiura M, Noguchi T (2008) Biochemistry 47:1024– 11030 P005 Influence of D1-H332, a ligand to the Mn4Ca-cluster, in the water oxidation mechanism of the oxygenevolving photosystem II enzyme Miwa Sugiura1, Yohei Ohno1, Fabrice Rappaport2, Hiroyuki Suzuki3, Takumi Noguchi3, Hidenori Hayashi1, Alain Boussac4 1 Cell-Free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan. 2 Université P. et M. Curie, CNRS UMR 7141, 13 rue P. et M. Curie, 75005 Paris, France. 3 Institute of Materials Science, University of Tsukuba, Ibaraki 305-8573, JP. 4 iBiTec-S, CNRS URA 2096, CEA Saclay 91191 Gif/Yvette, France. msugiura@chem.sci.ehime-u.ac.jp The active site for water oxidation in photosystem II goes through five oxidation states (S0–S4). The light-induced O2 evolution involves a Mn4Ca-cluster bound to at least seven amino acids of the D1 and CP43 polypeptides. The nature of the interactions of the Mn4Ca cluster with the surrounding amino acids is a key point to preserve the high driven force required to oxidize water. To study 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 the role of one of these ligands, the D1-H332, we analyzed purified PSII from T. elongatus in which this amino acid residue was replaced by either Gln or Ser. The effect of the mutation was investigated by a range of techniques like oxymetry, thermoluminescence measurements, time-resolved UV–visible absorption change, EPR and FTIR spectroscopies. Although the S3–S0 transition seemed kinetically unaffected, the O2 evolution activity was found to be 70–80% of that in the WT* (the WT* is a strain which expresses only one of the three variant copies encoding D1 protein [1]). Thermoluminescence measurements indicated that the thermodynamic properties of the S3 state were affected, i.e. the redox potential of S3 was decreased. This decrease could originate from the structural change detected by EPR in the S3 state of the H332 mutants. All the results will be discussed in the frame of the relationships between structural, kinetics and thermodynamic properties involved in the S3TyrZ + 2H2O to S0 + O2 transition. Reference 1. Sugiura M, Boussac A, Noguchi T, Rappaport F (2008) F Biochim Biophys Acta 1777:331–342 P006 Potent inhibitors of nitrous oxide reductase from Achromobacter cycloclastes Koyu Fujita, David Dooley Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717 USA. koyu@montana.edu Nitrous oxide reductase (N2OR) catalyzes the 2-electron reduction of N2O to N2 and H2O in the terminal step of denitrification. The crystallographic data show that N2OR is comprised two identical subunits and has two copper groups, a so-called dinuclear CuA and tetranuclear CuZ. Recent studies have demonstrated that fully reduced CuZ plays a key role in the catalytic cycle [1]; however, details of the mechanism of N2O reduction are not well understood. Although inhibition studies using small anions are a powerful approach to understand enzymatic mechanisms, few such investigations have been reported with N2OR. Turnover kinetics and spectroscopic studies of Achromobacter cycloclastes N2OR (AcN2OR) in the presence of azide, cyanide, fluoride and chloride, respectively, have been performed to help elucidate the catalytic mechanism. Data show that azide and cyanide are potent inhibitors of AcN2OR, and a slow, tight-binding model best described the inhibition. The data are consistent with some of these anions binding at the CuZ site (Fig. 1). Fig. 1 a Kinetics behavior of N2OR with respect to [N2O] in the presence of NaN3. b UV–vis titration of oxidized AcN2OR with NaN3 Reference 1. Chan JM, Bollinger JA, Grewell CL, Dooley DM (2004) J Am Chem Soc 126:3030–3031 J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 P007 A single methionine residue at the docking interface dramatically affects the interprotein electron transfer from cytochrome c to Cu-containing nitrite reductase Hiroyasu Koteishi, Masaki Nojiri, Kazuya Yamaguchi, Shinnichiro Suzuki Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan. koteishi@chem.sci.osaka-u.ac.jp Dissimilatory copper-containing nitrite reductase (CuNIR) catalyzes a one-electron reduction of NO2- to NO. CuNIR folds a trimeric structure and has two distinct copper centers in each monomer which are Type 1 Cu (T1Cu) and Type 2 Cu (T2Cu). T1Cu relays an electron from a donor protein to T2Cu, in which NO2- is reduced to NO. Recently, we have succeeded in determining the crystal structure of a protein–protein binary complex of CuNIR with its redox partner protein, cytochrome c551 (Cyt c551) from Achromobacter xylosoxidans, at 1.7 Å resolution. In the protein–protein docking interface between CuNIR and Cyt c551, Met135 of CuNIR locates at the center of the hydrophobic core region. To investigate the role of Met135 residue, site-directed mutagenesis was performed and kinetic analysis of electron transfer reaction between M135S mutant and Cyt c551 was carried out by stopped-flow techniques. The Ser mutation of Met135 dramatically affected the electron transfer rate constant. It was suggested that Met135 is important for the electron transfer between these proteins. Furthermore, we have determined the crystal structure of M135S CuNIR at 2.3 Å. The structural comparison between native and mutant NIR will be also discussed. P008 Systematic synthesis of heptadecanuclear manganese oxo clusters involving mixed valence Mn13 supercubane cores Ryoko Kubota, Yayoi Okui, Florina Catusanu, Takayuki Nakajima, Tomoaki Tanase* Department of Chemistry, Faculty of Science, Nara Women’s University, Nara 630-8506, Japan. tanase@cc.nara-wu.ac.jp Recently, manganese oxo clusters have attracted increasing attention with relevance to PSII OEC as well as nano-structured molecular devices such as single-molecule magnet (SMM). Using triscarboxylate Kemp’s triacid (H3kta) and terminal ligands L, we have systematically prepared novel heptadecanuclear manganese oxo clusters formulated as [Mn17O14(kta)6(L)4] [L = bpy (1), phen (2), 4,7-Ph2phen (3), dmf (4), etc.] (Fig. 1a), in which a mixed valence Mn13 supercubane core, 8+ II {MnIVMnIII 10Mn2 (l3-O)8(l5-O)6(kta)6} , is surrounded by four Mn(II) units terminated by L. A similar procedure with methanol used as L and S103 solvent gave another type of Mn17 oxo cluster, [Mn17O12(OMe)2(kta)6 II (MeOH)4] (5), which involves a two-electron reduced {MnIVMnIII 8 Mn4 8+ (l3-OMe)2(l3-O)6(l5-O)6(kta)6} supercubane core (Fig. 1b). Fig. 1 Structures of Mn17 clusters a 1 and b 5; MnIV (yellow), MnIII (violet), MnII (green) P009 Structure–function analysis of synthetic complexes obtained through inter-molecular disulfide bond between ferredoxin and ferredoxin-NADP+ reductase Yoko Kimata-Ariga, Yukiko Sakakibara, Takahisa Ikegami and Toshiharu Hase Institute for Protein Research, Osaka University, Osaka, Japan. a-yoko@protein.osaka-u.ac.jp Ferredoxin-NADP+ reductase (FNR) catalyses the conversion of NADP+ to NADPH using reduced ferredoxin (Fd) as an electron donor. Fd and FNR form a 1:1 complex, stabilized mainly by electrostatic interactions, for efficient electron transfer between their redox centers (2Fe–2S cluster and FAD, respectively) [1]. The relationship between electron transfer function and the interaction mode of Fd and FNR was investigated using Zea mays leaf Fd tethered to FNR by a disulfide bond in various configurations. The resulting Fd-FNR heterodimers showed a variety of efficiency for the intra-molecular electron transfer between Fd- and FNR domains. In order to investigate the interaction mode of Fd- and FNR domains of the heterodimers, NMR chemical shift perturbation analysis of Fd domain and absorption spectral analysis of flavin component of FNR domain were performed. Each two heterodimers with higher electron transfer efficiency (Fd21–FNR19 and Fd59– FNR19) and with lower efficiency (Fd21–FNR36 and Fd59–FNR36) were selected for the analyses. Both analyses showed that the interaction of Fd- and FNR domains in Fd21–FNR19 and Fd59– FNR19 is more similar to that of wild-type Fd:FNR complex than that of Fd21–FNR36 and Fd59–FNR36. However, small but significant differences in the FAD spectral changes and NMR chemical shift changes were observed between Fd21–FNR19 and Fd59– FNR19, although their electron transfer efficiency was almost the same, comparable to that of wild-type FNR with saturating concentration of Fd. Therefore, there seems to be a certain range of structural flexibility for attaining the maximum level of electron transfer between Fd and FNR. Reference 1. Kurisu G, Kusunoki M, Katoh E, Yamazaki T, Teshima K, Onda Y, Kimata-Ariga Y, Hase T (2001) Nat Struct Biol 8:117–121 123 S104 P010 Electron tunneling through mutant azurins on mixed-SAM gold electrodes Keiko Yokoyama1, Kyle M. Lancaster1, Yuling Sheng1, Nobuhumi Nakamura2, Hiroyuki Ohno2, Brian S. Leigh1, Katsumi Niki1, Jay R. Winkler1, John H. Richards1, Harry B. Gray1 1 Beckman Institute, California Institute of Technology, Pasadena California 91125, USA. 2Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. yokoyama@caltech.edu We have demonstrated that azurin (P. aeruginosa) exhibits particularly strong voltammetric responses on (alkanethiol + x-hydroxyl-alkanethiol) mixed-SAM electrodes. We report results for mutant azurins in which asparagine-47 was replaced by alanine, aspartic acid, lysine, arginine, leucine, threonine, serine, and glutamine. The N47D mutant on a mixed SAM exhibited a well-defined electrochemical response; N47T and N47S gave a weak signal; but the other five mutants showed no response. It is likely that the N47 side-chain carbonyl docks to the mixed SAM surface, providing a very favorable electron tunneling pathway to the copper via the N47–C112 hydrogen bond. We also examined the effect on the ET rates by mutation of the copper cysteine and methionine ligands. The ET rate constants for M121L azurin are similar to those of wild type; those for C112D are three orders of magnitude smaller; and those for C112D/M121L are two orders of magnitude smaller than those of wild-type at pH 7 (Fig. 1). J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 by proteolysis with pepsin/trypsin or chemically cleaved by cyanogen bromide to afford microperoxidases MPX (X = 8, 9, 11) and cyt c-65. Cobalt substitution into these frameworks provides another route to altering redox properties, and both iron and cobalt systems are being examined spectroscopically and electrochemically. Further, we are investigating the efficiency of these protein-based cobalt porphyrins as proton-reduction catalysts for solar-driven water splitting. P012 Construction of an artificial enzyme for olefin metathesis Clemens Mayer, Dennis Gillingham, Donald Hilvert Laboratory of Organic Chemistry, ETH Hönggerberg, Zürich, Switzerland. gillingham@org.chem.ethz.ch Olefin metathesis is a fundamentally new reaction in synthetic chemistry and it has quickly become established as one of the cornerstones in synthetic analysis and polymer chemistry. An underappreciated feature of olefin metathesis is its bioorthogonality. Current efforts to utilize this property are mired in the struggle to create an active and reliable water-soluble catalyst [1]. We describe our efforts to create an artificial metalloenzyme by covalently linking a metathesis catalyst to a protein scaffold. The new artificial metalloenzyme is optimized by mutagenesis to address selectivity problems in current olefin metathesis technology. This work is supported by a Marie-Curie International Incoming Fellowship to D.G. (IIF-AEOM). Reference 1. Jordan JP, Grubbs RH (2007) Angew Chem Int Ed 46:5152–5155 P013 Resonance Raman investigations of structure and dynamics of amino acid radicals as electron transfer intermediates in azurin Fig. 1 The proposed ET pathway and active site of P. aeruginosa azurin. (PDB code: 4AZU) Reference 1. Yokoyama K et al (2008) Inorg Chim Acta 361:1095–1099 P011 Heme peptides for catalytic hydrogen evolution from water Gretchen E. Keller, Jay R. Winkler, Harry B. Gray Beckman Institute, California Institute of Technology, Pasadena, CA 91125. gkeller@caltech.edu Cytochrome c is a small, well-characterized heme protein involved in electron transport. The wild-type protein has a reduction potential of +260 mV (vs. NHE); however, this potential is tunable via axial ligand (Met80) mutation and through metal substitution. We are investigating a series of cytochrome c variants where Met80 has been removed. The cytochrome c protein architecture can be stripped down 123 Hannah S. Shafaat, Brian S. Leigh, Michael J. Tauber, Judy E. Kim Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093 USA. judyk@ucsd.edu Tryptophan residues play a significant role in mediating biological electron transfer (ET) reactions, both as crucial elements of electron tunneling pathways through protein matrices and as radical intermediates that facilitate long-range electron transfer processes. Azurin, a blue copper electron transfer protein, contains a single native tryptophan residue in a hydrophobic pocket that may play a role in the natural ET pathway. Other non-native tryptophan residues in azurin have been shown to modulate ET rates through the protein. Here, we report on the direct reduction of the copper center from the natural tryptophan residue and present resonance Raman spectra of tryptophan radicals in the hydrophobic protein environment and in a solvent-accessible region. Our spectra show differential effects of pH and deuteration on the structures of these radicals in these distinct microenvironments. Additionally, the dynamics and quantitative mode displacements of the tryptophan excited state have been determined from resonance Raman scattering intensities, providing information on the internal reorganization energy and nuclear motions coupled to the electron transfer reaction. J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 Reference 1. Shafaat H, Leigh B, Tauber M, Kim J (2009) J Phys Chem B 113:382–388 P014 Time-resolved Resonance Raman study on structural relaxation process of cytochrome c oxidase following photolysis of carbonmonoxide Izumi Ishigami1, Satoru Nakashima2, Kyoko Shinzawa-Itoh1, Shinya Yoshikawa1,2, Takashi Ogura1,2 1 Department of Life Science. 2 Picobiology Institute, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan. rj07p002@stkt.u-hyogo.ac.jp Mitochondrial cytochrome c oxidase (CcO) reduces dioxygen to water, and transports protons from the negative to the positive spaces. CcO has four redox active centers. His376 and His378, ligating to hemes a3 and a, respectively, are the residues of helix X. According to X-ray crystallography, helix X exhibits conformational change upon redox-change and ligand binding. Conformational change of helix X must be important in the function of CcO, since it links heme a3 (the dioxygen reducing site) with heme a (the proton pump site). In the present study, we examined structural relaxation process at heme a3 from the carbonmonoxide (CO)bound to the equilibrium fully reduced states following photolysis of CO using time-resolved resonance Raman spectroscopy. The mFe–His mode appeared at 220 cm-1 at 10 ns after CO-photolysis, which was 6 cm-1 higher than that of equilibrium reduced state of 214 cm-1. The frequency remained unchanged until 100 ns and then showed downshift exponentially with time with a rate constant of 1.39106 s-1. The porphyrin m9 mode at 230 cm-1 of CO-bound heme a3 reflects the planarity of porphyrin. It exhibited intensity reduction of 20% at 10 ns and further 40% at 100 ns, and stayed constant at larger delay times, of the original intensity before CO-photolysis. The bending mode of vinyl or formyl substituent of heme a3 exhibited upshift from 435 to 436 cm-1 later than 5.3 ms after CO-photolysis. These results lead us to conclude that a sequential protein dynamics occurs as follows after CO-photolysis; (1) planarity of heme a3 decreases within 100 ns, (2) Fe–His band becomes weaker in about 1 ls and (3) heme a3 shifts horizontally so that the peripheral group suffers steric hindrance. P015 The role of type1 copper-containing N-terminal domain of hexameric nitrite reductase from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans. Saori Ikebuchi, Masaki Nojiri, Felicia Shirota, Daisuke Hira, Kazuya Yamaguchi, Shinnichiro Suzuki. Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan. ikebuchi@chem.sci.osaka-u.ac.jp Copper-containing nitrite reductase (CuNIR) catalyzes a one-electron reduction of NO2- to NO. CuNIR folds a trimeric structure with two S105 distinct Cu sites per a ca. 37-kDa monomer unit. The type 1 Cu site (T1Cu) buried within each monomer relays an electron from the redox partner protein to the catalytic type 2 Cu site (T2Cu), where NO2- is reduced to NO. Recently, the structure of a CuNIR from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans (HdNIR) has been reported, establishing the existence of a new family of CuNIR where an additional T1Cu (T1CuN) containing cupredoxin domain is located at the N-terminus [1]. To understand the role of this N-terminal domain and T1CuN, mutagenesis of the histidine ligand residue for T1CuN to glycine (H119G) has been performed and characterized spectroscopically and biochemically. In the UV–vis spectrum of H119G, the absorption band at 600 nm [that is characteristic of oxidation state T1CuN (Cu2+)] is lower than that of wildtype (WT), so it is thought that T1CuN of H119G is in the reduction state (Cu+). The NO2--reducing enzyme assays and stopped-flow experiments using cytochrome c550 (Cytc550) as a electron donor protein were also carried out, and the results show that H119G has higher activity than WT. These results suggest that the reduction of T1CuN affects the interaction between HdNIR and Cytc550 (Fig. 1). Fig. 1 UV–vis spectra of H119G and WT Reference 1. Nojiri M et al (2007) Proc Natl Acad Sci USA 104:4315 P016 A new resonance raman marker band of cytochrome c oxidase Miyuki Sakaguchi1, Kyoko Shinzawa-Itoh1, Shinya Yoshikawa1,2, Hiroshi Fujii3, Takashi Ogura1,2 1 Department of Life Science. 2 Picobiology Institute, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan. 3 Okazaki Institute Integrative Bioscience, Okazaki 444-8787, Japan. rj08w013@stkt.u-hyogo.ac.jp The redox change of heme a was proposed to drive proton pump in cytochrome c oxidase (CcO) [1]. The OH group of the hydroxyfarnesylethyl (HFE) substituent of heme a is hydrogen-bonded to S382 in its oxidized state. Upon reduction, the hydrogen-bond is cleaved and the OH group of S382 rotates 110° to make a cavity for proton collection. At the same time, the OH group of HFE substituent rotates 120°, which might cause a frequency shift of the vibrational mode associated with the HFE group. CcO in the reduced state gave a resonance Raman (RR) band at 1,247 cm-1 upon Soret excitation and it showed an upshift to 1,250 cm-1 in the oxidized state. It showed deuterium sensitivity. It 123 S106 J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 also showed an upshift to 1,253 cm-1 upon freezing at -15 °C. No other porphyrin in-plane vibrational mode showed such a significant shift upon freezing. RR spectra of bis-imidazole complex of Fe2+-2vinyl-4-hydroxymethyl deuteroporphyrin gave a Raman band at 1,249 cm-1, which exhibited deuterium sensitivity. The pattern of Raman difference spectrum (H2O–D2O) was similar to that of CcO. The results suggest that the band at 1,247 cm-1 of CcO is assignable to a vibrational mode associated with the OH group of HFE substituent probably of heme a. It could be a marker band to monitor the conformational change of the HFE group during proton pumping. Reference 1. Tsukihara T et al (2003) Proc Natl Acad Sci USA 100:15304– 15309 backed up by exquisite redox potential tuning among the molecular components in photosystem (PS) I and II. This aspect, however, is still far from being unraveled. Only the redox potentials of cytochrome (Cyt) b559, consisting of two protein subunits and a heme iron, and a plastoquinone QA have been measured among the electron transfer components of PS II (Fig. 1). The reported values of both Cyt b559 and QA redox potentials, however, exhibit heavy scatters of more than 100 mV probably due to the low accuracy of chemical titration that has traditionally been used. In the present study, we have tried to measure the redox potentials of Cyt b559 and QA precisely by spectroelectrochemistry using an optically transparent thin-layer electrode cell. Comparison of the redox potentials thus determined [1] with reported values sheds light to the redox properties which are buried in the previous measurement. P017 One-electron oxidized product of iron(III) porphyrinate that is diamagnetic Akira Ikezaki1, Hideyuki Tukada2, Mikio Nakamura1,3 1 Department of Chemistry, School of Medicine, Toho University, Tokyo 143-8540, Japan. 2 Graduate School of Integrated Science, Yokohama City University, Yokohama 236-0027, Japan. 3 Division of Chemistry, Graduate School of Science, Toho University, Funabashi 274-8510. Japan. ikezaki@med.toho-u.ac.jp Elucidation of the electronic structures of high-valent iron porphyrins is quite important because they play important roles in a biological system. One-electron oxidation of iron(III) porphyrinates gives three possible products, (1) iron(IV) porphyrin complexes such as Fe(TMP)(OCH3)2 and (Fe=O)(TMP), (2) high-spin or mixed highand intermediate-spin iron(III) porphyrin radical cations such as [Fe(TPP)Cl]+ and [Fe(TPP)]2+, and (3) low-spin iron(III) porphyrin radical cations such as [Fe(TPP)(HIm)2]2+. In this paper, we present the fourth case. Namely, a novel low-spin iron(III) porphyrin radical cation, [Fe(TMP)(tBuNC)2]2+, that is diamagnetic. The spectroscopic and magnetic data of this complex will be presented. N C N N N Fe N N OCH3 N N Fe N N N N Fe N N C N N N H [Fe(TMP)(OCH3)2] [Fe(TMP)(HIm)2]2+ S=1 S=1 S=0 (dxy)2(dxz, dyz)2(a2u)2 (dxy)2(dxz, dyz)3(a2u)1 (dxz, dyz)4(dxy, a2u)2 [Fe(TMP)(tBuNC)2]2+ References 1. Ikezaki A, Tukada H, Nakamura M (2008) Chem Commun 2257– 2259 2. Ikezaki A, Ohgo Y, Nakamura M (2009) Coord Chem Rev (in press) P018 Spectroelectrochemistry of electron transfer components in photosystem II Tadao Shibamoto1, Yoshinori Kuroiwa1, Yuki Kato1, Miwa Sugiura2, Tadashi Watanabe1 1 Institute of Industrial Science, University of Tokyo, Japan. 2 Cell-Free Science and Technology Research Center, Ehime University. Japan. watanabe@iis.u-tokyo.ac.jp Photosynthesis proceeds with an overall quantum yield of ca. 1.0 through many energy and electron transfer steps, and this must be 123 Reference 1. Shibamoto T, Kato Y, Watanabe T (2008) FEBS Lett 582:1490– 1494 2+ 2+ H N OCH3 Fig. 1 Electron transfer scheme of PS II P019 Redox potentials of chlorophyll a and pheophytin a in the electron transfer chain of oxygenic photosynthesis determined by spectroelectrochemistry Yuki Kato1, Akimasa Nakamura1, Miwa Sugiura3, Tadashi Watanabe1 1 Institute of Industrial Science, University of Tokyo, Japan. 2 Cell-Free Science and Technology Research Center, Ehime University. Japan. yukikato@iis.u-tokyo.ac.jp Oxygenic photosynthetic organisms convert photon energy to chemical free energy very efficiently in the primary process through lightinduced charge separation and subsequent electron transfers in photosystems (PS) I and II. Chlorophyll (Chl) a is a major constituent in PSs (ca. 40–200 depending on the type of PS and species), while a small number of Chl a and its derivatives play key roles in the reaction center. In PS I, a heterodimer of Chl a and its C132 epimer, Chl a0 , constitutes P700 that works as the primary electron donor, and monomeric Chl a works as the primary acceptor; in PS II, P680 that is considered as a homodimer of Chl a and pheophytin (Pheo) a, a Mg depleted derivative of Chl a, work as the primary electron donor and acceptor, respectively. Among them, the redox potentials of P700 and Pheo a have traditionally been investigated by chemical redox titration, and have been used to estimate those of the other components, since the P680 and Chl a potentials are too high and low to measure J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 directly, respectively. However, heavy scatters and non-negligible experimental errors exist in the reported potentials of P700 and Pheo a, rendering the electron transfer mechanism ambiguous. To overcome the drawbacks inherent to the titration, we have applied spectroelectrochemistry to potential determination. The precise values of P700 and Pheo a potentials thus determined are discussed in relation to the electron transfer mechanism (Fig. 1). S107 Recently, cytochrome c4A (PhCyt c4A, ca. 9kDa) was isolated from P. haloplanktis and spectroscopically characterized. Moreover, the intermolecular electron transfer from this heme–protein to PhNIR has been observed. Electron-transfer rate constants of PhNIR with PhCyt c4A were determined to be 3.3 9 104 M-1s-1 at pH 6.5 (25 °C) by the cyclic voltammetry method (Fig. 1). Fig. 1 Electron transfer scheme based on the redox potentials of the chain components P020 Computational study for the radical scavenging effects of carotenes Ching-Han Hu Department of Chemistry, National Changhua University of Education, Changhua, Taiwan. chingkth@cc.ncue.edu.tw Carotenes are an important type of molecules that occur naturally in plants and photosynthetic organisms. They are known to perform free radical scavenging and singlet oxygen quenching effects in biological tissues. Carotenes are also important plant pigments. They act as light harvesting antenna in the photosynthetic system, and protect the plant from the harmful effects caused by singlet oxygen and triplet chlorophyll. Recent progresses of our research group on the understanding of properties and reactivities of carotene species will be presented. In our research, the radical scavenging effects of carotenes were examined. Hydrogen abstraction and radical addition mechanisms were explored with quantum chemistry approaches. The addition mechanism was shown by our data to be the more thermodynamically favorable reaction path. P021 Intermolecular electron transfer reaction of c-type heme-containing copper nitrite reductase from Pseudoalteromonas haloplanktis TAC125 Ryosuke Ishikawa, Masaki Nojiri, Kazuya Yamaguchi, Shinnichiro Suzuki Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan. ryosuke@chem.sci.osaka-u.ac.jp The c-type heme-containing copper nitrite reductase(PhNIR), isolated from a psychrophilic bacterium, Pseudoalteromonas haloplanktis TAC125, has been structurally characterized. PhNIR folds a trimeric structure and has one c-type heme and two distinct copper centers in each monomer which are Type1Cu and Type2Cu. PhNIR accepts one electron from an external electron-donor protein and catalyzes the one-electron reduction of nitrite ion to nitrogen monoxide. Fig. 1 Cyclic voltammograms of PhCyt c4A (a) and after addition of PhNIR and 50 mM NO2- (b) P022 Low-frequency distortion modes of heme detected by femtosecond coherence spectroscopy Minoru Kubo1,3, Flaviu Gruia1, Abdelkrim Benabbas1, Alexander Barabanschikov1, William R. Montfort2, Estelle M. Maes2, Paul M. Champion1 1 Department of Physics, Northeastern University, Boston, MA 02115, USA. 2 Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 8572, USA. 3 Present Address: Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan. mkubo@sci.u-hyogo.ac.jp Resonance Raman spectroscopy has yielded significant information about the heme structures and dynamics of a variety of heme proteins through the Fe-ligand stretching bands and oxidation/spin marker bands of heme. This conventional spectroscopy, however, has the shortcoming that the low frequency region (\200 cm-1) is difficult to access because it is obscured by Rayleigh and quasi-elastic scattering. Femtosecond coherence spectroscopy (FCS), which is the recently advanced time-domain version of Raman spectroscopy, enables us to monitor low frequency Raman-active modes of a resonant chromophore in a protein. In this study [1], low frequency mode assignments have been made for iron porphine model compounds using FCS in combination with DFT calculations. FCS has also been applied to NP4, a NO-transport heme protein. We find a mode near 60 cm-1 that is conspicuously sensitive to the ruffling out-of-plane distortion of heme. Importantly, the band intensity of this mode depends quadratically on the magnitude of the ruffling distortion. To quantitatively account for this correlation, a new ‘‘distortion-induced’’ Raman enhancement mechanism that is uniquely relevant to low frequency soft modes is presented [1]. 123 S108 Reference 1. Kubo M, Champion PM et al (2008) J Am Chem Soc 130:9800 P023 Structure and reactivity of metal ion complexes of non-heme iron(IV)-oxo species Yuma Morimoto1, Hiroaki Kotani1, Pance Naumov1, Yong-Min Lee2, Wonwoo Nam2, Shunichi Fukuzumi1 1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, SORST, JST, Osaka 565-0871, Japan. 2 Department Chemistry, Division of NanoSciences, Ewha Womans University, Seoul 120-750, Korea. y-morimoto@chem.eng.osaka-u.ac.jp Metal ions play pivotal roles in biological electron-transfer (ET) systems such as photosynthesis and respiration. For example, the oxidation of water to dioxygen is catalyzed by Mn–oxo clusters with Ca2+ ion at the oxygen-evolving center (OEC) in photosystem II. We report herein the effects of redox-inactive metal ions on the structure and ET reactivity of non-heme iron(IV)–oxo complexes for the first time. Addition of redox inactive metal ions such as Sc3+ to a non-heme oxoiron(IV) complex ([(TMC)FeIV(O)]2+) resulted in formation of Sc3+ binding iron(IV)–oxo complex, which is confirmed by the X-ray crystallography as shown in Scheme 1. The ET properties of Sc3+ binding iron(IV)–oxo complex was compared to that of [(TMC)FeIV(O)]2+ [1]. The binding of Sc3+ enabled the two-electron reduction of [(TMC)FeIV(O)(Sc3+)]5+ by ferrocene, whereas only the one-electron reduction of [(TMC)FeIV(O)]2 occurred without Sc3+. Scheme 1 Reference 1. Lee Y.-M., Kotani H, Suenobu T, Nam W, Fukuzumi S (2008) J Am Chem Soc 130:434–435 P024 A systematic screening of polynuclear manganese complexes for water oxidation catalysis Denys Shevchenko, Anders Thapper, Magnus Anderlund, Stenbjörn Styring Department of Photochemistry and Molecular Science, Uppsala University, 751 20 Uppsala, Sweden. denys.shevchenko@fotomol.uu.se We have carried out a systematic screening of a series of known polynuclear manganese complexes, most of which are tetranuclear, for their ability to catalyze water oxidation and evolve oxygen. The compounds can be divided on four groups based on ligand types. The first group is complexes with mono- and bidentate ligands. The second and third ones consist of complexes based on tri- and more than 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 tridentate ligands, respectively. The fourth group includes polyoxometalate complexes. KHSO5 (oxone) and [Ru(bpy)3](ClO4)3 were used as oxidants. Oxygen evolution has been detected in four cases in the reaction with oxone and in one case in the reaction with [Ru(bpy)3](ClO4)3. In most cases formation of a fine brown precipitate was observed after or even before oxidant addition. Therefore, it is most likely that the starting complexes act as precursors for the in situ formation of the active species as result of solvolysis or oxidation reactions. It has been found that active species in the case of (n-Bu)4N[Mn4O2(PhCOO)7(pic)2] [1] (Hpic-2-picolinic acid) is a product of hydrolysis suggested to be hydrated manganese oxide. It shows a maximum oxygen evolution rate one order of magnitude higher (64.3 mMO2 s1 M-1 metal) than commercial manganese dioxide at the same conditions. Acknowledgment: This research was supported by the Swedish Energy Agency, the Knut and Alice Wallenberg Foundation and a Marie Curie International Incoming Fellowship (No. 236511) within the 7th European Community Framework Programme. Reference 1. Libby E, McCusker JK, Schmitt EA, Folting K, Hendrickson DN, Christou G (1991) Inorg Chem 30:3486 P025 Glutathione transferase: GSH activation mechanism proposal Daniel F.A.R. Dourado1, Pedro Alexandrino Fernandes1, Bengt Mannervik2, Maria João Ramos1 1 REQUIMTE/Departamento de Quı́mica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal. 2 Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, 75123 Uppsala, Sweden The cell detoxification mechanism of xenobiotic and endobiotic compounds follows a series of different steps. To begin with, toxic compounds are converted into strong electrophiles, by the mixedfunction oxidation activity of cytochrome P-450. Those electrophiles are subsequently transformed into more soluble and less toxic substrates, by conjugation with glutathione (GSH) due to the catalytic activity of glutathione transferases (GSTs), which are recognized by ATP-dependent transmembrane pumps such as Pglycoproteins and MRP family proteins, and consequently expelled from the cell. GSTs studies are of great importance since they have been implicated in the development of drug resistance in tumoral cells and are related to human diseases such as Parkinson’s, Alzheimer’s, atherosclerosis, liver cirrhosis, aging and cataract formation. In terms of structure GSTs can be homodimers or heterodimers having each monomer two active centers, a G-site pocket for glutathione (GSH) and an H-site pocket for the electrophilic substrate. When GSH binds to the G-site, the pKa of its thiol group drops 1.5 units promoting its deprotonation. This strong nucleophilic thiolate is now able to react with the electrophilic substrate, bounded in the H-site, building up a more soluble and less toxic compound. The nature of the residue that, behaving as a base, deprotonates the GSH thiol group is still unknown. Based on QM/ MM calculations we propose a mechanism for GSH activation with an overall free energy barrier consistent with the enzyme kinetics experimental studies. Reference 1. Dourado DF, Fernandes PA, Mannervik B, Ramos MJ (2008) Chemistry 14:9591–9598. J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 P026 Oxygen–oxygen bond formation pathways induced by Ru–Hbpp complexes Sophie Romain, Fernando Bozoglian, Xavier Sala, Antoni Llobet Institute of Chemical Research of Catalonia (ICIQ), Avinguda Paı̈sos Catalans 16, 43007 Tarragona, Spain, Departament de Quı́mica Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain. allobet@iciq.es The oxidation of water to molecular dioxygen is a reaction that takes place in the dark at the OEC-PSII. It is a very interesting reaction to be modeled from a bioinorganic perspective since it can give some hints regarding the potential mechanisms that operate in this natural system. On the other hand, it is of tremendous importance from an energetic perspective, since it is recognized to be the bottleneck for the development of commercial light harvesting devices for the photo production of H2 from water [1]. In 2004, we reported a new water oxidation catalyst {[RuII(trpy)(H2O)]2(l-bpp)}3+ [2] (trpy is 2,20 :60 ,200 -terpyridine, bpp is 2,6-bis (pyridyl)-pyrazolate) containing a pyrazolate bridging unit, whose structure is shown in the figure. We now report a thorough kinetic analysis combined with O18 labeling experiments that allows us to clearly elucidate the reaction mechanism, showing that is only intramolecular [3]. N N CuA centre is the primary electron acceptor from cytochromes c552 or c550. Heme a3 and CuB form the site for oxygen reduction. Our activity assays and binding studies reveal that oxidized yeast iso-1 Cc (YCc) remains bound to CcO. Moreover, YCc contains an exposed Cys opposite the docking site for CcO. The thiol can be used to anchor the protein directly to gold, promoting fast electron transfer [1]. YCc thus allows for the formation of a permanent, oriented substrate/enzyme complex on the electrode. Direct electrochemical control of the redox states of both Cc and CcO facilitates steady state as well as pre-steady-state electroenzymology. N N N Ru N Ru N O H N S109 N O H H H N References 1. Sala X, Rodriguez M, Romero I, Escriche L, Llobet A (2009) Angew Chem Int Ed (asap) 2. Sens C, Romero I, Rodrı́guez M, Llobet A, Parella T, BenetBuchholz J (2004) J Am Chem Soc 126:7798 3. Romain S, Bozoglian F, Sala X, Llobet A (2009) J Am Chem Soc 131:2768 P027 Yeast cytochrome c as sticky substrate for P. denitrificans cytochrome c oxidase: application as Co-immobilized mediator for voltammetry F.G.M. Wiertz1, O.-M. Richter2, B. Ludwig2, H.A. Heering1 1 Leiden University, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden, The Netherlands. 2 Goethe Universität, Institute of Biochemistry, Max-von Laue-Str. 9, 60438, Frankfurt am Main, Germany. f.wiertz@chem.leidenuniv.nl Paracoccus denitrificans cytochrome c oxidase (CcO) catalyses the oxidation of four cytochrome c (Cc) by oxygen. In addition, four protons are translocated from the cytoplasm to the periplasm, generating a proton electrochemical gradient across the cytoplasmic membrane: 3þ þ 2H2 O þ 4Hþ 4Cc2þ þ O2 þ 8Hþ cytoplasm ! 4Cc periplasm CcO is a four-subunit membrane enzyme, containing two heme groups (a, a3) and two copper centers (CuA, CuB). The binuclear Reference 1. Heering HA, Wiertz FGM, Dekker C, de Vries S (2004) J Am Chem Soc 126:11103–11112 P028 Storage of solar energy in chemical bonds using horseradish peroxidase Matthew R. Hartings1, Maraia Ener1, Jay R. Winkler1, Harry B. Gray1 1 Beckman Institute, California Institute of Technology, Pasadena CA 91125, USA. hartings@caltech.edu Horseradish peroxidase is a heme protein capable of oxidizing many small molecule substrates. During turnover, a ferryl species is generated (compound II) followed by the generation of a ferryl radical cation species (compound I), where the cation is localized on the porphyrin. Previous studies have found that it is possible to create compound II upon iron oxidation by a photoexcited Ru(bpy)3 molecule in solution [1]. This study focused on understanding the dynamic processes leading to the observation of compound II. Our current work is exploiting the photogeneration of compound II in order to turn over the substrate into product in the absence of molecular oxygen. These experiments are a very real attempt to mimic photosynthetic systems in their ability to convert solar energy into chemical bonds. Reference 1. Berglund J, Pascher T, Winkler J, Gray H (1997) J Am Chem Soc 119:2464–2469 123 S110 P029 Synthesis, characterization, and oxygen-evolving activity of dinuclear ruthenium complex with a Bis-tpa type ligand (6-hpa) Ryoko Sugiyama, Yutaka Hitomi, Takuzo Funabiki, and Masahito Kodera Doshisha University, Graduate School of Engineering, Kyoto 610-0321, Japan. dti0563@mail4.doshisha.ac.jp Green plants have developed complex catalysts for the dioxygen evolution via a four-electron oxidation of two water molecules. Development of catalysts capable of achieving the reaction is important in terms of modeling photosynthesis in green plants. Ruthenium-based catalysts show activity of water oxidation. The first example of this catalyst is [{Ru(bpy)2(H2O)}2O] (ClO4)4 [1] (bpy = 2,20 -bipyridine) in which two ruthenium ions are linked by a l-oxo bridge. Its turnover number is quite low due to the low stability. A practical water oxidation catalyst must have much better activity than this complex. This problem has been addressed by the design of ligands that increase the stability of the catalyst. In this work, We have synthesized a new dinuclear ruthenium complex [Ru2(H2O)2(OH)2(6-hpa)](ClO4)2 (1) to investigate dioxygen-evolving activity of this complex. The complex 1 was obtained as follows. A mixture of 6-hpa and [RuCl2(DMSO)4] in MeOH was refluxed for 8 h under Ar atmosphere. After concentration, NaClO4 was added to give a yellow precipitate. The solid was dissolved in an aqueous acetone, and AgClO4 was added to remove coordinated chloride ion, resulting in a deep green solution with white solid of AgCl. After filtration of the solid and concentration of the filtrate, 1 was isolated as a green solid. J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 describe about the details of crystal structures and electrochemical properties of these copper complexes. First, we synthesized [CuII(Sp)DTC]PF6 (1) and [CuII(aSp)DTC]ClO4 (2), and performed them X-ray single crystal structural analysis. When compared with these coordination structures, we found that the torsion angle of N2 and S2 coordination planes in 2 is larger and more approximate to orthogonal in Td than that of 1. Second, the cyclic voltammogram of 1 and 2 exhibited quasi-reversible redox processes of CuII/III and CuI/II, respectively. Taking into account these results, we discussed about formation of low-valent (1+) and high-valent (3+) copper centers in S-containing ligand fields with tetrahedral distortion (Fig. 1). Fig. 1 Crystal structure of [CuII(Sp)DTC]+ P031 Fine-tuning of functional and structural model complexes for the active site of copper-containing nitrite reductase Reference 1. Meyer TJ et al (1985) J Am Chem Soc 107:3855–3864 P030 Redox function of pseudo-tetrahedral copperdithiocarbamato complexes as a progressive analogue to type I copper center Tomoaki Toyama, Kotaro Yoshii, Takanori Inazumi, Tomohiko Inomata, Tomohiro Ozawa, Yasuhiro Funahashi, Hideki Masuda Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan. cgp11094@stn.nitech.ac.jp In blue copper proteins, type I copper sites are redox-active centers having pseudo-tetrahedral coordination with two His-imidazoles and Cys/Met-sulfur ligands. In this study, we synthesized new copperdithiocarbamato (DTC) complexes with a natural alkaroid, (-)sparteine (Sp), and its stereo isomer, a-isosparteine (aSp), as biomimetic model compounds of type I copper sites. Sp is able to enforce tetrahedral distortion around the metal center. In this report, we 123 Makoto Misoo1, Akiko Minami1, Yasushi Kai2, Shinnichiro Suzuki1, Shinobu Itoh3, Kazuya Yamagichi1 1 Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan. 2 Department of Environmental and Biotechnological Frontier Engineering, Fukui University of Technology, Fukui 910-8505, Japan. 3 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan. kazu@ch.wani.osaka-u.ac.jp Nitrite reductase (NIR), a key enzyme of denitrification, catalyzes the reduction of nitrite to nitrogen monoxide. Generally, copper-containing NIR possesses each of type 1 Cu (blue copper) and type 2 Cu (nonblue copper) per a monomer. The type 2 Cu site is bound by three His residues and one solvent water, which results in a distored tetrahedral geometry. We have reported that [Cu(Me2bpa)]2+ complex (Me2bpa: bis(6-methyl 2-pyridyl methyl) amine) is a good functional and structural model for the type 2 active site of NIR [1]. In this study, we prepared [Cu(XnbpaY)]2+ complexes with several substituents (X: H2, Me1H1, Me2; Y: H, Et, Bz, EtPh) and investigated the spectroscopic and electrochemical characterization, X-ray crystal structural analysis, and the catalytic activities of the NIR model complexes. The redox potentials and catalytic activities were obviously affected with the substituents. Moreover, the cation-p interaction between Cu2+ and phenyl ethyl substituent was observed (Fig. 1). These substituents are available for the fine-tuning of the catalytic activities and the electronic structures of NIR model complexes. J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 S111 P033 X-ray structure and function of peroxide bridge between Fe and Cu in the O2 reduction site of the fully oxidized cytochrome c oxidase Fig. 1 Reference 1. Yokoyama H, Yamaguchi K, Sugimoto M, Suzuki S (2005) Eur J Inorg Chem 2005:1435–1441 P032 Electron transfer reaction of porphyrin and porphycene complexes of Cu(II) and Zn(II) in acetonitrile Masahiko Inamo1, Kaori Aoki1, Toshimitsu Goshima1, Yohei Kozuka1, Yukiko Kawamori1, Noboru Ono2, Yoshio Hisaeda3, Hideo D. Takagi4 1 Department of Chemistry, Aichi University of Education, Kariya 448-8542, Japan. 2 Department of Chemistry, Ehime University, Matsuyama 790-8577, Japan. 3 Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka 819-0395, Japan. 4 Research Centre for Materials Science, Nagoya University, Nagoya 464-8602, Japan. minamo@auecc.aichi-edu.ac.jp The outer-sphere one-electron oxidation reaction of the Cu(II) and Zn(II) complexes of nonplanar 2,3,7,8,12,13,17,18-octaethyl5,10,15,20-tetra-phenylporphyrin (EOTPP) as well as the planar porphyrins and porphycenes by Cu2+ giving corresponding p-cation radicals was investigated in acetonitrile. The electron self-exchange rate constants between the parent porphyrin and porphycene complexes and their p-cation radicals were determined using the Marcus cross relation for the electron transfer reaction. The obtained rate constants are in the order of 109–1011 M-1 s-1 for the planar porphyrin and porphycene complexes and 104–106 M-1 s-1 for the nonplanar OETPP complexes at T = 25.0 °C. The relatively slow self-exchange reaction of the distorted porphyrin complexes was ascribed to the significant deformation of the complex associated with the oxidation reaction from the parent complex to the corresponding p-cation radical. Et Et Et Ph Ph N Et N Et M Et N N Et Ph Cu+ Et +• Ph Et Et Ph Et Cu2+ N N Et Ph M Et N Et N Ph Ph Et Et M = Cu(II), Zn(II) distorted more distorted Hiroshi Aoyama1,2, Kazumasa Muramoto3, Kyoko Shinzawa-Itoh3, Kunio Hirata4, Eiki Yamashita4, Tomitake Tsukihara3,4, Takashi Ogura3, Shinya Yoshikawa3 1 RIKEN SPring-8 Center, Sayo, Sayo 679-5148, Japan. 2 Graduate School of Pharmaceutical Science, Osaka University, Suita 565-0871, Japan. 3 Picobiology Institute, Department of Life Science, University of Hyogo, Kamighori, Akoh 678-1297, Japan. 4 Institute for Protein Research, Osaka University, Suita 565-0871, Japan. muramoto@sci.u-hyogo.ac.jp Cytochrome c oxidase (CcO) is the terminal oxidase of the respiratory chain embedded in mitochondrial and bacterial membrane. CcO catalyzes O2 reduction to H2O coupled to a proton pump across the membrane. The O2 reduction site consists of heme and copper atom (heme a3 and CuB). Electrons for O2 reduction are transferred from cytochrome c in the positive side space to the O2 reduction site via other metal centers (CuA and heme a). The protons for H2O formation are transferred from the negative side space via two hydrogen-bond network. The proton pumping pathway is proposed differently between mammalian and bacterial CcO. Recent X-ray structural analysis improved the resolution of bovine CcO structure. However, strong X-ray irradiation to CcO crystal induces reduction of the metal centers. Therefore, we performed the X-ray diffraction experiment minimizing X-ray irradiation effects by shortening irradiation time and using many crystals to examine the structure of the fully oxidized ‘‘as isolated’’ (without any reduction/oxidation treatment) bovine heart CcO. The X-ray structure showed a peroxide group bridging the two metal sites in the O2 reduction site (Fe3+–O-–O-–Cu2+). Physiological relevance of the present X-ray structural results will be discussed. Reference 1. Aoyama H, Muramoto K, Shinzawa-Itoh K, Hirata K, Yamashita E, Tsukihara T, Ogura T, Yoshikawa S (2009) Proc Natl Acad Sci USA 106:2165–2169 P034 Contrasting catalytic properties of the two uptake hydrogenases of Escherichia coli Michael Lukey1, Alison Parkin1, Frank Sargent2, Fraser Armstrong2 1 Department of Inorganic Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, England, UK. 2 College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. michael.lukey@balliol.ox.ac.uk The catalytic properties of the isoenzymes hydrogenase-1 (Hyd-1) and hydrogenase-2 (Hyd-2) of E. coli have been investigated by electrochemical methods. These proteins are known to be membrane-bound and to function in H2-uptake. Protein film voltammetry (PFV) experiments reveal that both enzymes are highly active in H2 oxidation, but only Hyd-2 also shows significant H2 production activity. While Hyd1 can function in 20% O2, H2 oxidation by Hyd-2 is completely inhibited by much lower O2 concentrations. Furthermore, after exposure to a transient O2 burst, Hyd-1 recovers rapidly and completely at a relatively high potential, whereas Hyd-2 recovers only partially and at a lower potential. H2 production by Hyd-2 is much less affected than H2 oxidation by the presence of O2, and H2 production activity can be 123 S112 J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 sustained for long periods of time in the presence of this gas. Hyd-2 is shown to be more sensitive than Hyd-1 to CO, a molecule known to be inhibitory to many other hydrogenase enzymes. The results are discussed in relation to the possible physiological roles of Hyd-1 and Hyd-2, and the varying conditions under which E. coli utilises H2. P035 Reactivity of Ru(IV)–oxo complexes derived from proton-coupled electron transfer of Ru(II)–aqua complexes Takahiko Kojima1, Yuichirou Hirai2, Yasuhisa Mizutani3, Kenichiro Ikemura4, Takashi Ogura4, Yoshihito Shiota5, Kazunari Yoshizawa5, Shunichi Fukuzumi2 1 Department of Chemistry, University of Tsukuba, Ibaraki 305-8751, Japan. 2 Department of Material Life Science, Osaka University, SORST(JST), Suita, Osaka 565-0871, Japan. 3 Department of Chemistry, Osaka University, Toyonaka, Osaka 5600043, Japan. 4 Graduate School of Life Sciece, University of Hyogo, Kouto, Hyogo 678-1297, Japan. 5 Institute for Materials Chemistry and Physics, Kyushu University, Moto-oka, Fukuoka 819-0395, Japan. kojima@chem.tsukuba.ac.jp Proton-coupled electron transfer (PCET) in which proton and electron are transferred simultaneously plays indispensable roles in biological redox reactions. In the course of water oxidation in photosynthesis, a high-valent manganese–oxo complex is proposed to be formed from a Mn–aqua complex via multistep PCET as a responsible species for the dioxygen evolution. Inspired by this process, Ru–aqua complexes have been converted to be isolable high-valent Ru–oxo complexes which can oxidize organic substrates. We synthesized novel Ru(II)–aqua complexes with use of tris(2-pyridyl- methyl)amine (TPA) [1] and its derivatives. They allowed us to have a series of Ru(IV)–oxo complexes bearing different spin states. Those complexes exhibited highly efficient and highly selective catalysis in oxidative conversion of organic substrates with similar rate constants and activation parameters. Details of mechanistic insights will be discussed. Oxidant RuII OH2 (S = 0) Oxidized Product O 2+ RuIV O (S = 0 or 1) Substrate N N OH2 RuII N N N OH2 [Ru(TPA)(H2O)2]2+ N 2+ O RuII N N OH2 [Ru(6-(COO–)-TPA)(H2O)2]+ Reference 1. Hirai Y, Kojima T, Mizutani Y, Shiota Y, Yoshizawa K, Fukuzumi S (2008) Angew Chem Int Ed 47:5772–5776 P036 Effects of axial ligands on the electron-transfer vs. proton-coupled electron-transfer reactions of non-heme oxoiron(IV) complexes Shunichi Fukuzumi1, Hiroaki Kotani1, Tomoyoshi Suenobu1, Seungwoo Hong2, Yong-Min Lee2, Wonwoo Nam2 1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, SORST, JST, Osaka 565-0871, Japan. 2 Department of Chemistry, Division of NanoSciences, Ewha Womans University, Seoul 120-750, Korea. fukuzumi@chem.eng.osaka-u.ac.jp 123 The effects of axial ligands on the electron-transfer reduction and the proton-coupled electron-transfer reduction of mononuclear non-heme oxoiron(IV) complexes were investigated using [FeIV(O)(TMC)]2+ (1) with various axial ligands X (1-X), where TMC is 1,4,8,11-tetramethyl- 1,4,8,11-tetraazacyclotetradecane and X is CH3CN (1-NCCH3), CF3COO- (1-OOCCF3), or N3- (1-N3), and ferrocene derivatives as one-electron reductants. As the binding strength of axial ligands increases, the one-electron reduction potentials of 1-X (Ered, V vs. SCE) are more negatively shifted in the order of 1-NCCH3, 0.39 V [ 1-OOCCF3, 0.13 V [ 1N3, -0.05 V. Rate constants of electron transfer from ferrocene derivatives to 1-X were analyzed in light of the Marcus theory of electron transfer to determine reorganization energies (k) of electron transfer [1]. The effect of the axial ligands is the deceleration of the electron-transfer rate in the order of 1-NCCH3 [ 1-OOCCF3 [ 1-N3. In sharp contrast to this, the rates of the proton-coupled electrontransfer reduction of 1-X are markedly accelerated in the presence of acid in the opposite order such as 1-NCCH3 \ 1-OOCCF3 \ 1-N3. Such contrasting effects of the axial ligands on the electron-transfer and proton-coupled electron-transfer reactions of non-heme oxoiron(IV) complexes are discussed in light of the counterintuitive reactivity patterns observed in the oxo-transfer and hydrogen-atom abstraction reactions by non-heme oxoiron(IV) complexes. Reference 1. Lee Y.-M., Kotani H, Suenobu T, Nam W, Fukuzumi S, J Am Chem P037 Photoinduced hydrogen production with artificial photosynthesis based on chlorophyll–carotenoid conjugated system Yutaka Amao, Yuko Maki, Yoshiko Fuchino Department of Applied Chemistry, Oita University, Oita 870-1192, Japan. amao@cc.oita-u.ac.jp Light-harvesting site in photosynthesis protein consists of Mg chlorophyll-a, b (MgChl-a, b) and carotenoid dye such as b-carotene. MgChl-a and b play an important role in photosynthesis such as light harvesting, the photoinduced electron transfer and so on. On the other hand, carotenoid dyes also have important functions such as the absorption of UV light, photo-protection of MgChl-a and b, and photosynthesis protein. In photosynthesis protein, MgChl-a, b and carotenoid dyes are assembled via the hydrogen bond, hydrophobic interaction and coordination bond, not covalently. Thus, MgChl-a, b and carotenoid dyes can be assembled using hydrophobic interaction of surfactant micellar as photosynthesis protein mimics. In this work, artificial photosynthesis system, anionic water-soluble carotenoid dye crocetin (kmax =536 nm) electrostatically immobilised onto the surface of cationic surfactant cetyltrimethylammonium bromide (CTAB) micellar including MgChl-a and b (Cro/MgChl), is prepared and applied to the photoinduced hydrogen production system with platinum nano-particle catalyst. The fluorescence at 680 nm due to MgChl-a and b is observed. However, the fluorescence at 572 nm due to crocetin is disappeared. In contrast, the fluorescence also is observed at 680 nm with excitation to absorption band of MgChl-a and b (660 nm). Moreover, the weak fluorescence at 680 nm is observed with 536 nm excitation in the CATB micellar including MgChl-a and b without crocetin (MgChl). These results indicate that the photoinduced energy transfer from the photoexcited state of crocetin to MgChl-a and b occurs. When the sample solution containing Cro/MgChl, methylviologen, NADPH and colloidal platinum was irradiated, the hydrogen production was observed. J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 P038 Electronic structure description of tris(dithiolene) complexes of molybdenum and tungsten Stephen Sproules1, Eckhard Bill1, Serena DeBeer-George2, Karl Wieghardt1 1 Max-Planck-Institut für Bioanorganische Chemie, Mülheim an der Ruhr, Germany. 2 Stanford Synchrotron Radiation Lightsource, Stanford, USA. sproules@mpi-muelheim.mpg.de Pterin-containing molybdenum and tungsten enzymes catalyze a variety of two-electron oxygen redox reactions vital to the global cycling of carbon, nitrogen and sulfur. The metal is coordinated by a bidentate dithiolene ligand that links it to the pyranopterin cofactor, and thus the protein. Molybdenum and tungsten tris(dithiolene) complexes have been utilized to model the active sites of these enzymes in order to investigate the role of the dithiolene as a redox active ligand in the absence of an oxo moiety. There has been recent controversy over the true electronic structure of these compounds, with the neutral complex being described as possessing either a Mo(V) [1] or Mo(IV) [2] center, with one or two oxidized ligands, respectively. We present here a systematic study of the three-membered electron transfer series [M(dithiolene)3]0/1-/2- (M = Mo, W), and the use of electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies in concert with theoretical calculations to assign oxidation levels to this electron transfer series. References 1. Kapre RR, Bothe E, Weyhermüller T, DeBeer George S, Wieghardt K (2007) Inorg Chem 46:5642–5650 2. Tenderholt AL, Szilagyi RK, Holm RH, Hodgson KO, Hedman B, Solomon EI (2008) Inorg Chem 47:6382–6392 P039 Cytochrome P450s involved in oxidative coupling reactions: intermediates involved in generation of the chromopyrrolic acid (CPA) scaffold of rebeccamycin by joint action of RebO and RebD Tatyana Spolitak, David P. Ballou Department of Biochemistry, University of Michigan, Ann Arbor, MI 48109, USA. dballou@umich.edu The cytochromes P450 participate in a wide variety both biosynthetic and biodegradative processes, usually by catalyzing monooxygenation. In addition to oxygenations, P450s also catalyze atypical P450 chemistry such as reductions, isomerizations, and oxidative couplings. This work presents experimental evidence for catalytic intermediates involved in the rebeccamycin biosynthetic pathway, which includes two P450-dependent reactions (RebD and RebP) that effect the carbon–carbon coupling of two modified tryptophans to form the rebeccamycin scaffold. Our results suggest that these oxidative coupling reactions involve the oxo-ferryl species (Cpd I) of RebD. We show that RebD can carry out peroxidase chemistry, implying that it can form Cpd I, thought to be a key oxidant in the coupling reaction of RebD [1]. In the presence of RebO to produce the modified tryptophan substrate (B), Cpd II was observed, suggesting that a nascent Cpd I had oxidized the substrate to form a tryptophan radical. The radical is suggested to form C, which in subsequent steps is coupled to form the rebeccamycin agycone. S113 Supported by NIH grant GM20877. Acknowledgment: We thank C. Walsh and A. Howard-Jones for providing plasmids that were used in this work. Reference 1. Howard-Jones AR, Walsh CT (2005) Biochemistry 44(48):15652– 15663. P040 Towards the understanding of His411–FeIV=O spectroscopic properties in ferryl intermediate of cytochrome c oxidase 1 O2 reaction: a theoretical QM/MM, MD approach Vangelis Daskalakis1, Stavros C. Farantos1,2, Victor Guallar3, Constantinos Varotsis2,* 1 Institute of Electronic Structure and Laser, Foundation for Research and Technology, Hellas, P.O. Box 1527, 711 10 Heraklion, Greece. 2 Department of Chemistry, University of Crete, P.O. Box 2208, 71003 Voutes, Heraklion, Greece. 3 Barcelona Supercomputing Center, Centro Nacional de Supercomputación and Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. vdas@her.forthnet.gr; varotsis@edu.uoc.gr Cytochrome c oxidase (CcO), found in the inner mitochondrial membranes or in many bacteria, catalyzes the four electron reduction of molecular oxygen to water. Four protons are pumped across the inner mitochondrial membrane, by CcO, attributing to the electrochemical gradient [1] needed by ATP-synthase. QM/MM and MD calculations are used to probe the spectroscopic characteristics of the ferryl intermediates in aa3 cytochrome c oxidase (CcO) from P. denitrificans. We link proton pump activity in CcO enzyme to m(FeIV=O) stretching vibrational frequency in a higher level of theory, than previously applied [2], and to the interesting d(His411– FeIV=O) bending vibration. We find that the His411–FeIV=O moiety vibrations become highly coupled depending on the protonation state of the heme a3 ring A propionate/Asp399 pair and we propose a mechanism of the resonance Raman enhancement of the d(His411–FeIV=O) bending vibration due to an 1:2 resonance phenomenon involving CuB motion towards heme a3. Implications of this CuB motion for the CcO mechanism of action will also be discussed. Proposed experiments can probe only the d(His411– FeIV=O) vibration in the 350–360 cm-1 lower frequency region of the spectrum, as MD calculations show that by exciting the FeIV=O bond we can chose to enhance either both m(Fe=O) and d(His411– Fe=O) or only the latter. References 1. Wikström MK (1977) Nature 266:271–272 2. Daskalakis V, Farantos SC, Varotsis C (2008) J Am Chem Soc 130(37):12385–12393 123 S114 P041 Directional electron transfer of four heme cytochrome c3 investigated by EQCM measurement Noriyuki Asakura, Hiromu Matsumoto, Takumi Tezuka, Ichiro Okura 1 Department of Bioenginerring, Tokyo Institute of Technology. nasakura@bio.titech.ac.jp A simultaneous measurement of electrochemistry and quartz crystal microbalance (EQCM) applied for monitoring of the cytochrome c3 and hydrogenase interaction. Cytochrome c3 has four hemes in one molecule and is the substrate for hydrogenase which catalyses both hydrogen evolution and uptake. Reduced cytochrome c3 donates electrons to hydrogenase and hydrogen evolution occurs. While oxidized cytochrome c3 accepts electrons from hydrogenase in the case of hydrogen uptake. The role of each heme in the electron transfer of cytochrome c3 is not still clarified. 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S101–S114 In this study, an electron-donating heme (electron exit) and an electron-accepting heme (electron entrance) were investigated by EQCM measurement. Cytochrome c3 was immobilized on a quartz crystal gold electrode and the immobilized cytochrome c3 redox was controlled by electrode potential. When the immobilized cytochrome c3 is reduced, hydrogen evolution complex is formed, and otherwise hydrogen uptake evolution is formed when the cytochrome c3 is oxidized. The complex interchange driven by cytochrome c3 redox was monitored by EQCM measurement. In order to investigate the electron entrance and exit in cytochrome c3, two types of cytochrome c3 immobilized electrode were prepared. One is that the heme I is faced toward electrode, and the other is that the heme IV is faced toward electrode. EQCM measurement for the two electrodes was carried out. The result shows that heme I is the electron entrance and that heme IV is the electron exit.